Glutathione-S-transferase (GST) fusion proteins consisting of the D, E and F domains of human (a ), (GST-hERa ) and rainbow trout (GST-rtER) estrogen receptors (ERs) were used to examine potential differences in binding affinities of estrogenic compounds. E2, OH-tamoxifen and DES exhibited similar binding to both proteins; however, there were significant differences in binding affinities for select compounds. Coumestrol bound with 13-fold lower affinity to GST-rtER than GST-hERa , while 4-t-octylphenol and o,p´-DDT bound with a 10-fold greater affinity to GST-rtER compared to GST-hERa . Molecular modeling and ligand docking tools were used to investigate the structural basis for these differences. 3-D chemical structures docked to a hERa crystal structure and a homology model of the rtER using AutoDock, identified L349 in hERa , and the corresponding residue, M315, in rtER as potentially contributing to differences in ligand preference. Both the wild type and the L349M mutant exhibited comparable Kd values for E2 (0.5 nM). The binding affinities of estradiol, OH-tamoxifen, DES, 4-t-octylphenol and o,p´-DDT were unchanged. However, the binding affinity of coumestrol was reduced 7-fold, comparable to that observed with rtER. This suggests that residue L349/M315 plays a central role in the observed differences in ligand binding between the human and rainbow trout ERs.